New ligand, N-(2-pyridylmethyl)aminoacetate, for use in the immobilised metal ion affinity chromatographic separation of proteins

A new chelating compound has been developed for use in the immobilised metal ion affinity chromatographic separation of proteins. The tridentate ligand, sodium N-(2-pyridylmethyl)aminoacetate (carbpyr), I, was prepared via a one-step synthesis from 2-picolylamine, 3 and then immobilised onto Sepharose CL-4B through the epoxide coupling procedure. The binding behaviour of the resulting IMAC sorbent, following chelation with Cu2+ ions to a density of 152 mu mol Cu2+ ions/g gel was characterised by frontal analysis experiments using horse heart myoglobin (HMYO) at pH 7.0 and pH 9.0. From the derived isotherms, the adsorption capacity, q(in), for the binding of HMYO to immobilised Cu2+-N-(2-pyridylmethyl)aminoacetate (im-Cu2+-carbpyr)-Sepharose CL-4B at these pH values was found to be 1.92 and 1.91 mu mol/g sorbent, respectively, whilst the dissociation constants K-D were 0.0092.10(-6) M and 0.0062.10(-6) M at pH 7.0 and pK 9.0, respectively, indicating that the HMYO-im-Cu2+-N-(2-pyridylmethyl)aminoacetate complex was more stable under alkaline conditions, although the binding capacity in terms of mu mol protein/g gel remained essentially unchanged. The selectivity features of the im-Cu2+-carbpyr-Sepharose CL-4B sorbent were further characterised in terms of the binding properties with several human serum proteins at pH 5.0, pH 7.0 and pH 9.0. (C) 1999 Elsevier Science B.V. All rights reserved.